An in vitro Study on the Role of Hepatitis B Virus X Protein C-Terminal Truncation in Liver Disease Development.

Hepatitis B virus X protein C-terminal 127 amino acid truncation is often found expressed in hepatocellular carcinoma (HCC) tissue samples. The present in vitro study tried to determine the role of this truncation mutant in the hepatitis B-related liver diseases such as fibrosis, cirrhosis, HCC, and metastasis. HBx gene and its 127 amino acid truncation mutant were cloned in mammalian expression vectors and transfected in human hepatoma cell line. Changes in cell growth/proliferation, cell cycle phase distribution, expression of cell cycle regulatory genes, mitochondrial depolarization, and intracellular reactive oxygen species (ROS) level were analyzed. Green fluorescent protein (GFP)-tagged version of HBx and the truncation mutant were also created and the effects of truncation on HBx intracellular expression pattern and localization were studied. Effect of time lapse on protein expression pattern was also analyzed. The truncation mutant of HBx is more efficient in inducing cell proliferation, and causes more ROS production and less mitochondrial depolarization as compared with wild type (wt) HBx. In addition, gene expression is altered in favor of carcinogenesis in the presence of the truncation mutant. Furthermore, mitochondrial perinuclear aggregation is achieved earlier in the presence of the truncation mutant. Therefore, HBx C-terminal 127 amino acid truncation might be playing important roles in the development of hepatitis B-related liver diseases by inducing cell proliferation, altering gene expression, altering mitochondrial potential, inducing mitochondrial clustering and oxidative stress, and changing HBx expression pattern.

HIV-1 Nef promotes ubiquitination and proteasomal degradation of p53 tumor suppressor protein by using E6AP.

Human Immunodeficiency Virus-1 (HIV-1) Nef promotes p53 protein degradation to protect HIV-1 infected cells from p53 induced apoptosis. We found that Nef mediated p53 degradation is accomplished through ubiquitin proteasome pathway in an Mdm2-independent manner. By GST pulldown and immunoprecipitation assays, we have shown that Nef interacts with E3 ubiquitin ligase E6AP in both Nef transfected HEK-293T cells and HIV-1 infected MOLT3 cells. The p53 ubiquitination and degradation was found to be enhanced by Nef with E6AP but not by Nef with E6AP-C843A, a dominant negative E6AP mutant. We show that Nef binds with E6AP and promotes E6AP dependent p53 ubiquitination. Further, Nef inhibits apoptosis of p53 null H1299 cells after exogenous expression of p53 protein. The p53 dependent apoptosis of H1299 cells was further reduced after the expression of Nef with E6AP. However, Nef mediated reduction in p53 induced apoptosis of H1299 cells was restored when Nef was co-expressed with E6AP-C843A. Thus, Nef and E6AP co-operate to promote p53 ubiquitination and degradation in order to suppress p53 dependent apoptosis. CHME3 cells, which are a natural host of HIV-1, also show p53 ubiquitination and degradation by Nef and E6AP. These results establish that Nef induces p53 degradation via cellular E3 ligase E6AP to inhibit apoptosis during HIV-1 infection.

Impact of length of replication competent genome of hepatitis B virus over the differential antigenic secretion.

Hepatitis B virus (HBV) genome consists of circular partially double stranded DNA of 3.2 kb size which gets converted into covalently closed circular DNA (cccDNA) during its life cycle. It then acts as a template for formation of pregenomicRNA (pgRNA) of 3.5 kb. Absence of appropriate animal models prompted a need to establish a better in vitro culture system to uncover the propagation and survival mechanisms of the virus. There is scarcity of data to represent the significance of varying length of replication competent viral genome on the secretion of viral secretory proteins/antigens and in turn on the overall effects on the accomplishment of the viral life cycle. The present study was undertaken to ascertain a suitable replication competent construct in which the viral life cycle of HBV with varying clinical relevance can be studied efficiently. Two constructs (pHBV 1.3 and pHBV 1X) of different sizes were used to transfect hepatoma cells and consequently the secretory antigens were monitored. In vector free approach (pHBV 1X), 3.2 kb viral DNA is directly transfected in the culture system whereas in vector mediated approach more than full length of viral genome is cloned in a vector (pHBV 1.3X) and transfected to obtain a 3.5 kb pgRNA intermediate. HBV secretes two important antigens; HBsAg and HBeAg. HBsAg is a hallmark of infection and is the first to be secreted in the blood stream whereas HBeAg is a secretory protein and remains associated with the viral replication. The construct pHBV 1.3X referring to as more than full length, by virtue of being capable of undergoing transcription without the synthesis of cccDNA intermediate (unlike the clinical situation where an intermediate step of cccDNA synthesis is an essential component to initiate the viral life cycle) appears to be better system for studying viral life cycle in in vitro culture system. The reasons could be assigned to the fact that as low as 100 ng of viral DNA was shown to quantify the replicative phenotypes with this construct. The better efficiency of this construct at prima facie, appears to be mediated through the significantly higher levels of pgRNA transcript during the viral life cycle.

The host cell ubiquitin ligase protein CHIP is a potent suppressor of HIV-1 replication.

Human immunodeficiency virus-1 (HIV-1) Tat is degraded in the host cell both by proteasomal and lysosomal pathways, but the specific molecules that engage with Tat from these pathways are not known. Because E3 ubiquitin ligases are the primary determinants of substrate specificity within the ubiquitin-dependent proteasomal degradation of proteins, we first sought to identify the E3 ligase associated with Tat degradation. Based on the intrinsic disordered nature of Tat protein, we focused our attention on host cell E3 ubiquitin ligase CHIP (C terminus of HSP70-binding protein). Co-transfection of Tat with a CHIP-expressing plasmid decreased the levels of Tat protein in a dose-dependent manner, without affecting the corresponding mRNA levels. Additionally, the rate of Tat protein degradation as measured by cycloheximide (CHX) chase assay was increased in the presence of CHIP. A CHIP mutant lacking the U-box domain, which is responsible for protein ubiquitination (CHIPΔU-box), was unable to degrade Tat protein. Furthermore, CHIP promoted ubiquitination of Tat by both WT as well as Lys-48-ubiquitin, which has only a single lysine residue at position 48. CHIP transfection in HIV-1 reporter TZM-bl cells resulted in decreased Tat-dependent HIV-1 long-terminal repeat (LTR) promoter transactivation as well as HIV-1 virion production. CHIP knockdown in HEK-293T cells using CRISPR-Cas9 led to higher virion production and enhanced Tat-mediated HIV-1 LTR promoter transactivation, along with stabilization of Tat protein. Together, these results suggest a novel role of host cell E3 ubiquitin ligase protein CHIP in regulating HIV-1 replication through ubiquitin-dependent degradation of its regulatory protein Tat.

USP7 deubiquitinase controls HIV-1 production by stabilizing Tat protein.

Deubiquitinases (DUBs) are key regulators of complex cellular processes. HIV-1 Tat is synthesized early after infection and is mainly responsible for enhancing viral production. Here, we report that one of the DUBs, USP7, stabilized the HIV-1 Tat protein through its deubiquitination. Treatment with either a general DUB inhibitor (PR-619) or USP7-specific inhibitor (P5091) resulted in Tat protein degradation. The USP7-specific inhibitor reduced virus production in a latently infected T-lymphocytic cell line J1.1, which produces large amounts of HIV-1 upon stimulation. A potent increase in Tat-mediated HIV-1 production was observed with USP7 in a dose-dependent manner. As expected, deletion of the USP7 gene using the CRISPR-Cas9 method reduced the Tat protein and supported less virus production. Interestingly, the levels of endogenous USP7 increased after HIV-1 infection in human T-cells (MOLT-3) and in mammalian cells transfected with HIV-1 proviral DNA. Thus, HIV-1 Tat is stabilized by the host cell deubiquitinase USP7, leading to enhanced viral production, and HIV-1 in turn up-regulates the USP7 protein level.